The application of recombinant DNA technology to industrially important organisms such as Streptomyces and related actinomycete genera requires efficient gene cloning and transformation procedures.
Transformation of Streptomyces and related genera is well known in the art. Transformation of Streptomyces and related genera using standard transformation procedures requires that the recipient cells be enzymatically converted to protoplasts. Several drawbacks to the protoplast transformation methods have, however, impeded the wide application of recombinant DNA technology in many species of Streptomyces and related genera. First, the protocols required for efficient transformation vary greatly, and subtle procedural details often need to be worked out before productive cloning experiments can proceed. Compare the transformation procedure of Matsushima et al., 1985, J. Bacteriol. 163: 180-185 with that of Yamamoto et al. 1986, J. Antibiotics 39: 1304-1313. Second, most Streptomycetes produce restriction endonucleases (see Cox and Baltz, 1984, J. Bacteriol. 159: 499-504 and Lomovskaya et al., 1980, Microbiol. Rev. 44: 206-229) that can decrease the efficiency of phage infection and plasmid transformation. See Matsushima and Baltz, 1985, J. Bacteriol. 163: 180-185; Chater and Wilde, 1980, J. Gen. Microbiol. 116: 323-334; Chater and Wilde, 1976, J. Bacteriol. 128: 644-680; and Chater and Carter, 1978, J. Gen. Microbiol. 109: 181-185. The problem caused by restriction endonucleases is often compounded by the rigid procedural requirements for efficient uptake of plasmid DNA and protoplast regeneration. Physiological conditions for cell growth that might minimize the expression of restriction endonucleases often inhibit efficient uptake of DNA, plasmid replication, and protoplast regeneration.
A bacteriophage-mediated transduction system circumvents many of the problems encountered in protoplast transformation procedures. In a transduction system, the transducing DNA can be packaged into phage particles, which can attach and inject DNA, and thus, transduce intact cells, thereby avoiding the need to prepare and regenerate protoplasts. Intact cells can tolerate a broader range of culture conditions, especially temperature of incubation, better than protoplasts. A transduction system can circumvent the problems encountered with host restriction systems, because some host restriction systems may become less active as the temperature of incubation varies from that of optimal growth. In addition, a transduction system can be used simply to overwhelm host restriction systems, for by raising the multiplicity of infection (m.o.i.), one increases the amount of transducing DNA introduced into a cell. Finally, phage-mediated transduction can be used to transform different strains of the same species and even other species and genera. Plasmid-mediated transformation systems, however, are often limited by the narrow host range of the transforming plasmid. The significant advantages inherent in a transduction system are presently limited in their useful scope of applications by the necessity of using a low m.o.i. to prevent lysis of the recipient cells by intact phages.
The present invention provides DNA compounds which were isolated from phage FP43 and which confer a plaque inhibition pin phenotype on cells comprising the pin sequence. Thus, host cells comprising the pin sequence are transducible at high m.o.i.